Noscapine Crosses the Blood-Brain Barrier and Inhibits Glioblastoma Growth

Vol. 10, 5187–5201, August 1, 2004 Clinical Cancer Research 5187

Jaren W. Landen,1 Vincent Hau,8 rier, interfere with microtubule dynamics, arrest tumor cell Mingshen Wang,2 Thomas Davis,8 Brian Ciliax,2 division, reduce tumor growth, and minimally affect other dividing tissues and peripheral nerves, warrant additional Bruce H. Wainer,3 Erwin G. Van Meir,4,5,6 investigation of its therapeutic potential. Johnathan D. Glass,2 Harish C. Joshi,1 and David R. Archer7 INTRODUCTION Departments of 1Cell Biology, 2Neurology, 3Pathology, Patients diagnosed with glioblastoma (WHO grade IV) 4Neurosurgery, 5Winship Cancer Institute, and 6 7 have a median survival of 9–12 months despite surgical resec- Hematology/Oncology, and the AFLAC Cancer Center and Blood tion, radiation therapy, and/or chemotherapy (1, 2). The infil- Disorder Service, Emory University School of Medicine, Atlanta, Georgia; and 8University of Arizona, Department of Pharmacology, trative nature of astrocytic tumor growth rarely allows complete Tucson, Arizona surgical resection, and more than 90% of tumors recur within 2 cm of the primary tumor site. Postoperative radiotherapy pro- longs survival, but the prognosis is still less than 2 years. ABSTRACT Intrinsic chemoresistance and poor penetrance of drugs through The opium alkaloid noscapine is a commonly used an- the blood-brain barrier remain significant challenges for the titussive agent available in Europe, Asia, and South Amer- chemotherapeutic treatment of gliomas (3). Given the limited ica. Although the mechanism by which it suppresses cough- efficacy of existing therapy, even when combined, there is a ing is currently unknown, it is presumed to involve the considerable need to direct research efforts to develop more central nervous system. In addition to its antitussive action, effective treatments for brain tumors. noscapine also binds to tubulin and alters microtubule dy- Malignant gliomas develop in part as a result of genetic namics in vitro and in vivo. In this study, we show that mutation(s) in checkpoint genes resulting in deregulation of the noscapine inhibits the proliferation of rat C6 glioma cells in cell cycle. Abrogation of the G -S checkpoint is a frequent event ␮ 1 ؍ vitro (IC50 100 M) and effectively crosses the blood-brain in the development of gliomas (4–7), implying a role for cyclin- barrier at rates similar to the ones found for agents such as dependent kinases cyclin-dependent kinase 4/6 and their morphine and [Met]enkephalin that have potent central catalytic partners and D-type cyclins that are required for pro- nervous system activity (P < 0.05). Daily oral noscapine gression through the G1-S phases of the cell cycle. The cyclin- treatment (300 mg/kg) administered to immunodeficient dependent kinase/cyclin D complex is inhibited in response to mice having stereotactically implanted rat C6 glioblasoma DNA damage or inadequate cell growth by p16INK4 and CIP/ into the striatum revealed a significant reduction of tumor KIP, resulting in the activation of the G -S checkpoint and arrest volume (P < 0.05). This was achieved with no identifiable 1 of normal cells in G (8–13). Homozygous deletions of G -S toxicity to the duodenum, spleen, liver, or hematopoietic 1 1 checkpoint genes have been found in 41% of glioblastomas, cells as determined by pathological microscopic examination suggesting that checkpoint mutations may contribute to the of these tissues and flow cytometry. Furthermore, noscapine uncontrolled cell proliferation of glioblastoma (14). Other mu- treatment resulted in little evidence of toxicity to dorsal root tations have also been well documented including mutations in ganglia cultures as measured by inhibition of neurite out- p53 (15) or in the retinoblastoma gene (16), each of which is growth and yielded no evidence of peripheral neuropathy in found in nearly one-half of all gliomas. animals. However, evidence of vasodilation was observed in Our laboratory has identified a microtubule-interacting noscapine-treated brain tissue. These unique properties of noscapine, including its ability to cross the blood-brain bar- chemotherapeutic agent that overcomes many of the limitations associated with other tubulin-binding drugs. This agent, noscapine, is an antitussive opium alkaloid that lacks sedative, eu- phoric, analgesic, and respiratory depressant properties (17). Received 2/24/04; revised 5/6/04; accepted 5/10/04. The precise mechanism for the antitussive effects of noscapine Grant support: NIH (H. Joshi, E. Van Meir, J. Glass), American is unknown, although it appears centrally mediated. Noscapine Cancer Society (H. Joshi), the University Research Committee (H. can reduce electrically induced cough, characteristic of drugs Joshi), the Beat Leukemia Jill Andrews Fund (D. Archer), and Brain affecting the autonomic nervous system (18), and radiolabeled Tumor Foundation for Children (D. Archer). noscapine binds the central nervous system (19, 20). Cough The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked suppression was the only pronounced pharmacological effect of advertisement in accordance with 18 U.S.C. Section 1734 solely to noscapine known for more than 30 years. In the last 5 years, we indicate this fact. demonstrated that noscapine: (a) binds to tubulin and alters its Note: H. Joshi and D. Archer contributed equally to this work. conformation and assembly properties; (b) interferes with mi- Requests for reprints: David R. Archer, Emory University School of Medicine, Department of Pediatrics, Emory University School of Med- crotubule dynamics both in vitro and in living cells; (c) arrests icine, 1462 Clifton Road, Room 466, Atlanta, GA 30322. Phone: a variety of mammalian cells in mitosis and targets them for (404) 727-1378; Fax: (404) 727-4859; E-mail: [email protected]. apoptosis; and (d) inhibits growth of murine thymoma cells,

human breast carcinoma, and melanoma cells in mice by induc- at room temperature, and visualized using immunofluorescence. ing polyploidy and apoptosis (21–23). Furthermore, in contrast To visualize DNA, tubulin-stained cells were further incubated to other microtubule-interacting agents such as paclitaxel, no- with 20 ␮g/ml propidium iodide (Boehringer Mannheim) for 30 codazole, and vinblastine, noscapine modifies microtubule dy- min before washing with PBS. Cells were subsequently namics without affecting total tubulin polymer mass in reconsti- mounted on coated glass slides and analyzed using confocal tuted systems and without altering the steady-state monomer/ microscopy for microtubule morphology, DNA content, and the polymer equilibrium of microtubule assembly in cells (23). number of cells in mitosis (at least 100 cells were examined per In this study, we show that glioma cell treatment with condition). In a separate experiment, to examine whether noscapine induces polyploidy. Noscapine-treated cells undergo noscapine-treated cells go through multiple rounds of DNA excessive DNA synthesis and atypical nuclear divisions in the synthesis, cells plated as described above were treated with absence of cytokinesis, resulting in multinucleated cells. We noscapine for 72 h. After 24 h, 30 ␮M bromodeoxyuridine further show that noscapine crosses the blood-brain barrier and (BrdUrd; Sigma Chemical Co.) was added for the remaining 48 h. inhibits the growth of subcutaneous (s.c.) and intracranially Cells were fixed and stained with anti-BrdUrd antibody (1:200 (i.c.) implanted rat C6 glioma cells in immunocompromised dilution; Boehringer Mannheim) and stained with FITC-labeled mice without apparent toxicity to organs with rapidly prolifer- secondary antibody (1:100; Jackson), mounted on glass slides, and ating tissues or induction of neurological symptoms. analyzed for BrdUrd incorporation by confocal microscopy. Flow Cytometric Analysis of Cell Cycle Status. Cell MATERIALS AND METHODS cycle status was determined by measuring cellular DNA content Mice and Cell Lines. Eight-week-old athymic female after staining with propidium iodide by flow cytometry (21). mice (nu/nu) were purchased from the National Cancer Institute Cells (1 ϫ 104) were plated on 10-cm dishes and incubated for (Bethesda, MD). The rat C6 glioma cell line (American Type 24 h before the addition of 0, 50, 250, 500, or 1000 ␮M Culture Collection) was maintained in DMEM supplemented noscapine in 1% DMSO for 0, 6, 12, 24, 48, 72, or 96 h. with 10% fetal bovine serum and passaged no more than 10 Staurosporine (100 nM), a well-known cytotoxic agent, was used times. Primary glial cells were isolated as follows. Cells from as a positive control. In an independent study, cells were treated the mouse subventricular zone of C57BL/6 mice were dissected with noscapine at the same dosages and durations specified as under a dissecting microscope, manually dissociated using a above, washed three times with PBS at 37°C, and allowed to flame polished pipette, and grown in DMEM containing 20% fetal bovine serum. Glial cells used for experiments were iden- recover for 96 h in fresh medium without noscapine. Cells from tified as cells that contain glial fibrillary acidic protein, an both experiments were removed with trypsin, collected, washed intermediate filament subunit found exclusively in glial cells. twice in ice-cold PBS, fixed overnight in 70% ethanol at Ϫ ϫ These cells did not express neuronal markers such as Tuj-1, an 20°C, and centrifuged at 1000 g for 10 min. Cells were then ␮ antibody that is specific for neuronal ␤-tubulin. resuspended in 30 l of phosphate/citrate buffer [0.2 M Cell Density Assay. Cell proliferation was determined Na2HPO4/0.1 M citric acid (pH 7.5)] and incubated with pro- ␮ ␮ by the WST-1 tetrazolium salt assay (Boehringer Mannheim), pidium iodide (20 g/ml) and RNase A (20 g/ml) in PBS for which quantifies the amount of formazan dye formed when 30 min. The propidium iodide fluorescence was measured using tetrazolium salt is cleaved by cellular mitochondrial enzymes a Becton Dickinson flow cytometer. Data were analyzed using present in viable cells. Cells were plated at a density of 1 ϫ Winlist software (Verity Software House, Topsham, ME). 103/well in 96-well microtiter plates in 0.2 ml of culture me- In Vitro Bovine Brain Microvessel Endothelial Cell As- dium. Cells were allowed to adhere overnight and then incu- say. Bovine brain microvessel endothelial cells were isolated bated with 0, 0.1, 1, 2, 10, 50, 100, or 1000 ␮M noscapine (97% from the cerebral cortex as described previously (24) on poly- purity; Aldrich; 100ϫ stock in DMSO) for 0, 12, 24, 48, 72, or carbonate membrane filters. In brief, bovine brain microvessel 96 h. The final concentration of DMSO in medium never ex- endothelial cells were isolated from the gray matter of the ceeded 1%. Five hours before the end of the specified incubation bovine cerebral cortex by enzymatic digestion followed by periods, 50 ␮l of WST reagent were added to the cells. At the subsequent centrifugations and seeded into primary culture. end of the incubation, cell density was estimated by measuring Polycarbonate membranes (13 mm; pore size, 3.0 ␮m; diffusion the absorbance of the colored formazan reaction product at 450 area, 0.636 cm2) were placed in tissue culture dishes (100 mm; nm using a microtiter plate reader (Molecular Devices Ltd., Corning, Corning, NY) and coated with rat-tail collagen and Crawley, West Essex, United Kingdom). bovine fibronectin (Sigma). Isolated brain microvessel endothe- Tubulin, DNA, and Bromodeoxyuridine Staining. Rat lial cells were seeded onto the prepared tissue culture dishes at C6 glioma cells and primary glial cells were cultured on poly- a density of 5 ϫ 104 cells/cm2 in a culture medium consisting of L-ornithine-coated glass coverslips and allowed to adhere for 45% MEM, 45% Ham’s F-12 nutrient mixture (Life Technolo- 24 h. To examine how noscapine affects microtubule morphol- gies, Inc., Grand Island, NY), 10 mM 4-(2-hydroxyethyl)-1- ogy and DNA content, noscapine was dissolved in DMSO, and piperazineethanesulfonic acid (pH 7.4), 13 mM sodium bicar- cells were then incubated at 37°C with 0, 25, 50, 250, 500, and bonate, 10% plasma-derived equine serum, 100 mg/ml heparin, 1000 ␮M noscapine for 24, 48, 72, or 96 h. Cells were fixed in 100 mg/ml streptomycin, 100 mg/ml penicillin G, 50 mg/ml methanol at Ϫ20°C for 5 min, incubated with anti-␣-tubulin polymyxin B, and 2.5 mg/ml amphotericin B (Sigma Chemical

antibody (DM1A; 1:500 dilution; Amersham Biosciences) for Co.). The cells were cultured at 37°C with 5% CO2. Medium 2 h at room temperature, washed and incubated with a goat was replaced on the 3rd day after seeding, and then every 2 days antimouse secondary antibody (1:200 dilution; Jackson) for 1 h until confluent monolayers were formed (10–14 days). Conflu-

ence was determined by inspecting the areas around the poly- paraformaldehyde in phosphate buffer. At necropsy, the follow- carbonate membranes with an inverted microscope. ing tissues were taken for analysis: spleen; duodenum; liver; Bovine brain microvessel endothelial cell monolayers cul- sciatic nerve; sural nerve; dorsal and ventral roots; and brain. tured on a polycarbonate membrane were placed in a Side-Bi- Before perfusion, blood from the heart was taken for a complete Side diffusion cell (Crown Glass Co., Somerville, NJ) contain- blood count using a complete blood count instrument (CDC ing 3 ml of continuously stirred physiological assay buffer (122 Technologies, Oxford, CT), and bone marrow was removed

mM NaCl, 3.0 mM KCl, 1.2 mM MgSO4,25mM NaHCO3, 0.4 from the right femur and tibia bones for WBC analysis using a mM K2HPO4, 1.4 mM CaCl2,10mMD-glucose, and 10 mM 25-gauge needle. Brain weight was obtained upon sacrifice for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) on each both s.c. and intracranial tumor groups. On day 21 (15 days of side at 37°C (Fig. 5). At time 0, noscapine (500 ␮M) was added noscapine treatment), animals in the s.c. tumor group (n ϭ 30) to the donor chamber, and 100-␮l aliquots were removed from received one final dose of noscapine (n ϭ 15) or vehicle the receptor chamber at various time points (15, 30, 60, 90, and solution (n ϭ 15) 2 h before sacrifice to determine noscapine 120 min) and stored for high-performance liquid chromatogra- concentrations in the brain at the reported half-life of noscapine phy (HPLC) analysis. An equal volume of assay buffer was (26). Animals in the s.c. group were sacrificed by cervical added to replace the aliquots removed. [14C]sucrose (10.44 dislocation, and the brains were retained for HLPC detection of Ci/mmol; NEN Research Products, Boston, MA), a molecule noscapine as described below. that does not cross the membrane, served as a negative control Determination of Noscapine Concentration in Animal and [Met]enkephalin (DPDPE; Tyr-Gly-Gly-Phe-Met), which is Tissues by High-Performance Liquid Chromatography. known to permeate the blood-brain barrier, was used as a Two h after the final noscapine administration (n ϭ 15) or positive control. Background leakiness was monitored and cor- vehicle solution (n ϭ 15), animals were anesthetized and then rected for by determining the levels of [14C]sucrose in the sacrificed by cervical dislocation. This timing was selected samples via scintillation spectrometry (efficiency, 93% for based on the reported half-life of noscapine (154 min; Ref. 27). [14C]sucrose; Beckman LS 50000 TD counter; Beckman Instru- Blood was collected before animal sacrifice directly from the ments Inc., Fullerton, CA). Passage of the test solute across the heart and centrifuged, and plasma was removed and stored at in vitro blood-brain barrier monolayer was determined by re- Ϫ80°C for HPLC analysis of noscapine and its metabolites. verse phase-HPLC as described previously (25). Reverse phase- Brains were removed without perfusion; meninges and exterior HPLC values were obtained in moles and used to determine blood vessels were dissected including the middle cerebral permeability coefficients found by using the following equation: artery, the arteries forming the circle of Willis, superior saggital ϭ ϫ ϫ PC X/(A t Cd) where PC is the permeability coefficient sinus, and the transverse sinus. Dissected brains were homoge- (cm/min), X is the amount of substance in moles in the receptor nized and centrifuged to remove cell debris, and supernatants chamber at time t (min), A is a constant diffusion area (0.636 were collected and stored at Ϫ80°C for HPLC analysis. HPLC 2 cm ), and Cd is the concentration of the substance in the donor analyses were performed in a double-blind fashion according to chamber (in mol cmϪ3). a previously published method (28). In brief, samples were In Vivo Tumorigenicity Assays. Immediately before analyzed on a reverse-phase-HPLC system consisting of a WISP surgery, rat C6 glioma cells were washed twice with PBS and 710B Autoinjector, two model 6000A Solvent Delivery Pumps, placed into serum-free DMEM medium. Thirty anesthetized Automated Gradient Controller (Waters Associates, Milford, 8-week-old athymic nude mice received 1 ϫ 103 rat C6 glioma MA), LC-65T Detector/Oven (210 nm; Perkin-Elmer, Norwalk, cells stereotactically implanted into the left striatum (coordi- CT), 3390A Integrator (Hewlett-Packard Co., Avondale, PA), nates: anterior-posterior, ϩ2.5; medial-lateral, ϩ3.5; dorsal- and a 218TP54 column (4.6 ϫ 250 mm; Vydac, Hesperia, CA). ventral, Ϫ2 mm; from the Bregma). C6 glioma cells in a volume Samples were eluted using a linear gradient of acetonitrile ␮ of 2 l were slowly injected over a time span of 15 min (for against 0.1 M NaH2PO4 buffer (pH 2.4). The flow rate was illustration, see Fig. 7A). Due to the age of the animals, the maintained at 1.5 ml/min, and the column temperature at 40°C. ϭ ϭ Ϫ needle easily penetrated the skull. Sham-operated animals (n The capacity factor was defined as follows: k (tr to)/to 15) received an identical intracranial injection of serum-free where tr is the retention time of the retained peak and to is the medium alone. After injection, the needle track was sealed with retention time of an unretained peak. bone wax, and the incision was closed with Ethicon staples Image Analysis. Brains from animals that received i.c. (Endo Surgery, Inc.). An independent group of 30 animals injections were fixed by perfusion as described above and received 1 ϫ 106 rat C6 glioma cells in a volume of 0.2 ml s.c. sectioned into 1-mm-thick slices using a brain matrix (for illus- into the right flank. Six days after s.c. injection when s.c. tumors tration, see Fig. 8B; Ted Pella, Redding, CA). Each 1-mm-thick were palpable or six days after stereotactic injection, animals section was then embedded in paraffin blocks maintaining were divided into two groups (n ϭ 15/group). One group re- proper orientation so that the anterior-most side would be sec- ceived noscapine hydrochloride by daily gavage [300 mg/kg tioned first. A single 5-␮m section was then cut from each block

dissolved in de-ionized water (dH2O; pH 4.5)], and the other and stained with H&E for three-dimensional reconstruction. group received the vehicle solution alone by gavage (dH2O; pH Slides were coded, and each section was captured at an identical 4.5). Tumor volumes were recorded biweekly for animals in the magnification using a digital camera (SPOT camera; Diagnostic s.c. group. On day 21 (15 days of noscapine treatment), animals Instruments Inc., Sterling Heights, MI). Tumor cells were iden- with intracranial tumors were anesthetized with 4% chloral tified because they stained with greater intensity than surround- hydrate and then perfused intracardially with phosphate-buff- ing normal striatal cells as shown in Fig. 7A (arrow). The ered-saline followed by 2.5% gluteraldehyde and 2.5% differences in staining intensity were detected using AIS image

analysis software (Imaging Research, Inc., St. Catherine’s concentration 1%) was added. Cultures were monitored and Heights, Ontario, Canada). The system was calibrated using a imaged on 0, 4, 8, and 10 days post noscapine treatment. The micrometer and selecting the appropriate number of pixels equal diameter of the circular halo of neurites was measured on the to 1 mm. Tumor cross-sectional area was determined by using initial day of noscapine exposure (day 0), and on days 4, 8, and an auto-sampling tool, which bases its selection on staining 10. Axonal survival was quantified by the longest remaining intensity. The autoscan tool uses an algorithm to select the target axon and the area of the remaining dorsal root ganglion halo region based on H&E-staining intensity differences between the (Fig. 10B). The axonal length was measured from the center of selected target gray value and background gray value identified. the halo to the visible distal ends of the axon in the periphery of The autoscan feature searches in concentric circles seeking a the halo. Halo areas were calculated by tracing the outside continuous boundary. This boundary is defined by an average circumference of the remaining culture halo. Because there was threshold level of staining intensity that circumscribes the target variability in the physical characteristics of individual cultures, area pixel by pixel, radiating outward from the first darkly each dorsal root ganglion served as its own control by normal- stained pixel selected. Adjacent tumor regions in the same izing data at days 4, 8, and 10 to the condition before noscapine section were identified as separate targets. This procedure en- exposure. Data were analyzed as a percentage change from day abled us to quantify infiltrative tumor regions consisting of 0 before noscapine treatment. Normalized data were then ex- normal and tumor tissue within a boundary because normal tissue had lower than threshold-staining intensity and thus was excluded from the cross-sectional tumor area measurements. The autoscan feature also excluded open spaces such as vessel lumens (for illustration, refer to Fig. 7, C and D). Tumor regions were verified by manual microscopic inspection by a pathologist (B. H. W.). The three-dimensional tumor volume was computed using cross-sectional tumor area ϫ 1 mm (the distance between 5-␮m sections). If more than one tumor target site was present in a cross-sectional tumor area, targets were added for that section. The blind code was broken after all of the sections had been scanned and tumor volumes obtained. Toxicity Evaluation. After animal perfusion and sacrifice on day 21, liver, duodenum, and spleen were sectioned, stained with H&E, and analyzed by two pathologists (B. H. W. and D. L. D.) for microscopic evaluation. Bone marrow was removed before fixation from the femur and tibia bones and analyzed by flow cytometry following antibody lineage-markers: CD3 (T cells); B220 (B cells); MAC-1 (macrophages); and Gr-1 (granulocytes; PharMingen, San Diego, CA). Cells were also incubated with 20 ␮g/ml propidium iodide to determine the percentage of dead cells. Sciatic nerve, dorsal root ganglion, dorsal and ventral roots, and sural nerves were removed from four animals from each group, embedded in plastic, cut at 1 ␮m, and stained with 0.5% toluidine blue for microscopic analyses. Sections were evaluated blindly for evidence of sensory and motor neuropathies by a neuropathologist (J. D. G.). Dorsal Root Ganglion Cultures and Evaluation of Neu- ropathy. Dorsal root ganglion neurons were cultured as described previously (29). In brief, dorsal root ganglions were dissected from newborn mice. Ganglia were transferred into L-15 medium (Life Technologies, Inc.), separated from roots and connective tissue sheaths, pooled, dissociated, and washed twice with PBS (pH 7.4). Dorsal root ganglions were then plated

(five per dish) in MEM supplemented with 1% N2 (Life Tech- nologies, Inc.), 10 ng/ml 7S nerve growth factor (Sigma), and 1.4 nML-glutamine (Sigma) and incubated at 37°Cina5% carbon dioxide atmosphere. Next, cultures were permitted to Fig. 1 Dose and time effects of noscapine on normal and tumor glial mature for 5 days to allow a lush halo of neurites around the cells in vitro. A, noscapine inhibits cell viability of rat C6 glioma in vitro explants to develop. Neuritic extensions were allowed to pro- in a dose-dependent manner. Noscapine exposure (250 ␮M; arrow) for ceed to evaluate the effect of noscapine on established neurites 72 h inhibited cell viability of rat C6 glioma cells (F) by 50%. Primary glial cells (ࡗ) were almost one-half as sensitive to noscapine (IC ϭ as opposed to the effect on primary neurite outgrowth. After 5 50 500 ␮M). B, kinetics of noscapine treatment on cell viability. Incubation days in the culture medium, the medium was changed and 25, with 250 ␮M noscapine for 24, 48, 72, and 96 h. F, rat C6 glioma cells; 50, or 250 ␮M noscapine or DMSO vehicle solution alone (final ࡗ, primary glial cells.

amined for statistical significance by ANOVA, with post-test resulted in a significant inhibition of glioma cells while primary correction for multiple comparisons (29). cells were nearly unaffected, we selected this dose to examine how this concentration affects cell viability over time (24–96 h; Fig. RESULTS 1B). We conclude that 250 ␮M noscapine exposure for 72 h is an Noscapine Inhibits Rat C6 Glioma Cell Proliferation. optimal dose to inhibit C6 glioma cell viability without signifi- To determine whether noscapine could inhibit glioma cell growth cantly reducing primary cell viability. in vitro, we chose the aggressive and rapidly dividing rat C6 glioma Noscapine Exposure Causes Abnormal S-Phase Re- cell line. Using the tetrazolium salt (WST-1) cell viability assay entry and Results in Excessive DNA Accumulation. The (see “Materials and Methods”), we generated a dose-response mechanism of the decreased cell viability observed in C6 cells curve by incubating cultures of rat C6 glioma cells and primary exposed to 250 ␮M noscapine for 72 h was evaluated by ana- mouse glial cells (as normal tissue control) with noscapine for 72 h lyzing the cell cycle distribution by flow cytometry with pro- and observed a dose-dependent inhibition of cell viability (Fig. 1A). pidium iodide (Fig. 2). Cultures of C6 cells or primary murine

Noscapine inhibited the viability of rat C6 glioma cells with an IC50 glial cells were treated with noscapine, fixed, and stained with of 250 ␮M at 72 h. Primary glial cells were less sensitive, having an the DNA intercalating fluorescent dye propidium iodide. Un- ␮ IC50 of 500 M (at 72 h). Manual counts of cell numbers verified treated C6 cells (Fig. 2C) and primary murine glial cells (Fig. this finding (not shown). Because 250 ␮M noscapine exposure 2A) exposed to vehicle alone (1% DMSO) had normal cell cycle

Fig. 2 Noscapine induces polyploidy in rat C6 glioma cells. Rat C6 glioma cells and murine primary glial cells were treated with 250 ␮M noscapine in DMSO or vehicle solution alone (DMSO) for 72 h and then fixed and stained with the DNA-binding fluorescent dye propidium iodide (PI) and with BrdUrd (BrdU). A, 72% of vehicle-treated primary glial cells contain 2N DNA content (peak in green), and 14% of cells contain 4N DNA content (peak in blue). Approximately 50% of primary cells incorporated BrdUrd (inset in green) compared with the nuclear staining shown in red. B, 24% of primary cells exposed to 250 ␮M noscapine for 72 h had 2N DNA content. Note the increase in the number of cells (37%) with 4N DNA content after noscapine exposure (peak in blue). Fifty percent of noscapine-treated primary cells incorporated BrdUrd (insets). C, vehicle-treated glioblastoma cells had a DNA content similar to vehicle-treated primary cells with 69 and 18% of cells containing 2N and 4N DNA content, respectively. BrdUrd-positive vehicle-treated glioma cells were observed in approximately 60% of cells. D, noscapine-treated glioma cells contained 1% of cells with 2N DNA content and 9% of cells with 4N DNA content. Note the position of the peak showing cells with abnormal ϳ8N-16N DNA content (yellow peak). This phenomenon is not seen in untreated C6 glioma cells (C) or in primary glial cells (A and B). Bar ϭ 5.0 ␮m.

profiles with approximately 70% of cells in G0-G1 phase with cumulate DNA by inappropriately reentering multiple rounds of 2N DNA content (Fig. 2, left peaks in green) and 16% of cells S phase, immunofluorescence was performed to detect BrdUrd

in G2-M phase containing 4N DNA content (Fig. 2, right peaks incorporation (Fig. 2, inset in green), a thymidine analog incor- in blue). In primary glial cells, noscapine exposure (250 ␮M for porated into cells during DNA synthesis (S phase). Nuclear

72 h) resulted in a decrease to 24% of G0-G1 phase cells with 2N staining with propidium iodide (Fig. 2, inset in red) was used to DNA content and an increase of cells in G2-M with 4N DNA detect all nuclei. Anti-BrdUrd staining was apparent in about content to 37% (Fig. 2B). In contrast, only 1% of rat C6 glioma 50% of the primary glial cells treated with vehicle alone (Fig. cells contained 2N DNA content, and 9% of cells contained 4N 2A, inset in green) or with noscapine (Fig. 2B, inset in green). DNA (Fig. 2D, blue peak) content after 72 h of continuous Anti-BrdUrd staining was also present in approximately 50% of noscapine exposure. In addition, 44% of cells contained 8N-16N C6 cells treated with vehicle (Fig. 2C, inset in green), indicating DNA (Fig. 2D, yellow peak). Primary glial cells (Fig. 2B) did that cell division occurred during the course of the experiment. not accumulate enhanced DNA content at identical doses, but In contrast, manual cell counts revealed that 97% of C6 cells rather, we observed reversible mitotic arrest after continuous incubated with noscapine for 72 h incorporated BrdUrd under noscapine exposure for up to 6 h (not shown). Flow cytometric these conditions, indicating that most cells were in S phase. data suggest that noscapine exposure causes 4N DNA accumu- These cells showed multiple discrete BrdUrd-positive micro-

lation in primary glial cells, suggesting G2-M arrest; whereas it nuclei (Fig. 2D, inset in green and nuclear staining in red) and results in 8N-16N DNA accumulation in C6 cells, suggesting had DNA content between 8N and 16N, suggesting that noscap- continuous DNA synthesis. ine-treated cells were undergoing multiple rounds of DNA rep- To determine whether C6 cells exposed to noscapine ac- lication in the absence of cytokinesis resulting in cells with

Fig. 3 Noscapine exposure results in multinucleated cells with abnormal mitotic figures in rat C6 glioma cells. Double-labeling immunofluorescence is shown with an anti-␣-tubulin antibody that stains microtubules in green and propidium iodide nuclear staining that is shown in red. Noscapine- treated rat C6 glioma cells have large, abnormal, multilobed nuclei and intact microtubule arrays (E); and mitotic figures, when observed, were abnormal with multiple microtubule asters and misaligned chromosomes (F). These effects were not apparent in noscapine-treated primary glial cells (B and C) or in vehicle-treated primary glia (A) or vehicle-treated rat C6 glioma cells (D). Note that noscapine exposure did not alter microtubule morphology in primary glial cells (B) or in rat C6 glioma cells (E). Bar ϭ 5.0 ␮m.

multiple nuclei. These features were not seen in primary cells (Fig. 2, A and B) or in vehicle-treated glioma cells (Fig. 2C). Noscapine treatment does not perturb the morphology of microtubule arrays and results in multinucleated glioma cells with abnormal mitoses. To examine microtubule morphology, cellular microtubule arrays were observed by immunofluorescence (Fig. 3). We used an antibody against ␣-tubulin (Fig. 3, green) to stain microtubules and the DNA-specific stain, propidium iodide (Fig. 3, red), to stain chromosomes. In vehicle- treated C6 glioma and primary glial cells, microtubule arrays were localized throughout the cytoplasm of interphase cells (Fig. 3, A and D). Microtubule arrays in vehicle-treated cells appeared similar to those in untreated control cells (not shown), indicating that the vehicle solution (DMSO final concentration was less than 1% in cell medium) had no effect on microtubule Fig. 4 Measurement of mitotic figures in glioblastoma and primary arrays. Microtubule morphology of interphase primary glial glial cells. Noscapine preferentially arrests glioma cells in mitosis. A cells in the presence or absence of 250 ␮M noscapine for 72 h time course of mitotic index is shown after noscapine exposure for 12, ␮ was similar (Fig. 3, compare A and B). Mitotic figures of normal 24, 48, 72, and 96 h. Noscapine treatment (250 M) resulted in an increase in the number of mitotic figures observed in a time-dependent glial cells were not visibly affected by noscapine, as shown by manner up to 24 h. Mitotic figures were observed in 68% of noscapine- chromosomes properly aligned on the metaphase (Fig. 3C). In treated primary glial cell cultures at 24 h (ࡗ). In contrast, only 36% of contrast, as already observed in Fig. 2, C6 glioma cells treated noscapine-treated rat C6 glioblastoma cells were observed in mitosis at with noscapine revealed large, abnormal, multiple nuclei (Fig. 24h(F). The number of cells observed arrested in mitosis after 48, 72, 3E). Their microtubule structure was similar to that of vehicle- or 96 h of noscapine exposure decreased in a time-dependent manner with 42% primary glial cells and 20% glioma cells arrested at 48 h and treated cells (Fig. 3, compare E with D). However, the spindle 12 and 5%, respectively, at 72 h. At 96 h, approximately 5% of both cell structure of mitoses observed in noscapine-treated glioma cells types were observed in mitosis. In the absence of noscapine, the number were abnormal with misaligned chromosomes and multiple mi- of cells in mitosis at any given time was approximately 5% (छ and E, crotubule asters (Fig. 3, compare F with C). These data suggest primary glia and rat C6 glioma, respectively). that noscapine treatment causes abnormal mitoses and accumulation of micro-nuclei in rat C6 glioma cells, whereas cultures of primary glial cells do not accumulate DNA, and normal mitoses are observed when exposed to noscapine. Noscapine was deposited at a concentration of 500 ␮M in the donor Noscapine Exposure Causes Increased Mitotic Arrest. chamber, and aliquots were removed from the receiver chamber at To quantify the number of cells in M phase of mitosis in the 15, 30, 60, 90, and 120 min. The noscapine concentration was presence or absence of noscapine, cells were treated with 250 determined by HPLC, and the apparent permeability coefficient (in ␮M noscapine for incubation periods ranging from 0 to 96 h and cm/min) calculated as described in “Materials and Methods.” Pas- then fixed and stained with an anti-␣-tubulin antibody and sage of noscapine across the barrier was determined by the con- propidium iodide, and the number of cells in mitosis counted centration detected by HPLC in the donor chamber compared with (Fig. 4). Noscapine treatment (250 ␮M) resulted in an increase in the receiver chamber. Noscapine was detected in the receiver the number of mitotic figures observed in a time-dependent chamber at a concentration of 10.21 ␮M (0.2%) after 15 min that manner up to 24 h. Mitotic figures were observed in 68% of increased to 96.6 ␮M after 120 min (19%; Fig. 5B). Noscapine was primary glial cell cultures at 24 h. This is a significant increase found to cross the simulated blood-brain barrier with a permeability from approximately 5% observed in vehicle-treated cells at any coefficient of 21.7 ϫ 10Ϫ4 cm/min. This rate is 31.8% more time point. In contrast, only 36% of noscapine-treated C6 cells efficient than morphine (14.8 ϫ 10Ϫ4 cm/min), an opiate known to were observed in mitosis at 24 h. The number of cells observed possess lipophilic character and permeate the barrier (Fig. 5B; P Յ arrested in mitosis after 48, 72, or 96 h of noscapine exposure 0.05; Student’s t test), and similar to the positive control, [Met]en- decreased in a time-dependent manner. This might be due to kephalin (DPDPE; 24.24 ϫ 10Ϫ4 cm/min). Although this model drug inactivation/degradation (reported half-life ranges from 1.7 does not account for drug metabolism that occurs in vivo, it pro- to 4.5 h; Ref. 27). These data are compatible with the hypothesis vides evidence that noscapine efficiently crosses the blood-brain that noscapine arrests normal and tumoral glial cells in M phase barrier compared with other agents known to permeate well. of the cell cycle. Although noscapine-treated glioma cells may Next, we determined whether noscapine was transported initially arrest in mitosis, they overcome the M-phase block and across the blood-brain barrier in vivo. We homogenized and cen- reenter multiple rounds of DNA synthesis. trifuged whole brains of noscapine-treated animals and then deter- Noscapine Crosses the Blood-Brain Barrier. To exam- mined noscapine concentration by HPLC in supernatants. The ine the ability of noscapine to cross the blood-brain barrier, we used average noscapine concentration obtained from brain homogenates a well-characterized in vitro assay (illustrated in Fig. 5A). The rate of noscapine-treated animals was 18.2 Ϯ 3.7 ␮M (ϮSD). The at which noscapine can cross a layer of cultured brain microvas- animals used for this study were not perfused, so the values ob- cular endothelial cells separating a donor and receiver chamber was tained include the amount of noscapine present in brain tissue and determined and compared with known permeant (morphine and the vascular network. These results are also consistent with the [Met]enkephalin) and nonpermeant ([14C]sucrose) molecules. radioactive data describing noscapine accumulation in rat brain

Fig. 5 Measurement of noscapine diffusion across an in vitro blood-brain barrier model. A, schematic of in vitro blood-brain barrier model depicting the donor chamber in which noscapine was directly added (right chamber) and the receiver chamber (left chamber) from which aliquots were taken at 15, 30, 60, 90, and 120 min and noscapine concentration was determined. Cham- bers were separated by a layer of bovine brain microvessel endothelial cells (BBMEC) cocultured with astrocytes on a polycarbonate membrane. B, permeability coefficients (P.C. value) derived from measurements taken from the receiver chamber aliquots and analyzed for noscapine concentration by HPLC analysis. Permeability coefficient values of noscapine compared with an opiate, morphine, or [Met]enkephalin, a compound known to permeate the barrier well, are shown in the table. Permeability of the negative control, [14C]sucrose was subtracted from each condition.

(19). Our in vitro and in vivo results suggest that noscapine is vehicle-treated animals was 2.56 Ϯ 1.62 g compared with capable of efficiently crossing the blood-brain barrier. 0.79 Ϯ 0.44 g removed from noscapine-treated animals. Noscapine Inhibits Rat C6 Glioma Growth in Vivo. To To demonstrate that noscapine can treat gliomas in their examine the ability of noscapine to inhibit the growth of tumors orthotopic brain location despite the blood-brain barrier, we next in vivo, we first injected rat C6 glioma cells s.c. into immuno- injected 1 ϫ 103 C6 cells i.c. into the striatum of immunocom- deficient mice. 15 days of noscapine administration beginning 6 promised mice (n ϭ 30). Noscapine daily by gavage (300 days after s.c. implantation of 1 ϫ 106 C6 glioma cells signif- mg/kg; n ϭ 15) or vehicle solution alone (n ϭ 15) was admin- icantly inhibited tumor growth (Fig. 6, P Յ 0.01; Student’s t istered to animals for 15 days beginning on day 6, and animals test). On day 21, s.c. tumor volume was reduced by 60% in the were euthanized 21 days after tumor implantation. The daily noscapine-treated group compared with the vehicle-treated noscapine dosage of 300 mg/kg (corresponding to approxi- group (1510 Ϯ 237 and 3739 Ϯ 586 mm3, respectively; n ϭ 12 mately six times the in vitro concentration) was chosen based on ␮ and 11 respectively). As a result of the large s.c. tumors, overall noscapine solubility and the favorable IC50 of 250 M at 72 h. animal body weight significantly increased in untreated animals Intracranial tumor volumes were analyzed as follows: perfused (mean weight at the time of sacrifice of vehicle-treated animals brains were cut into 1-mm-thick sections (Fig. 7B) and embed- was 28.61 Ϯ 2.02 g, and the mean weight of noscapine-treated ded into paraffin. The first 5-␮m section from each block was animals was 23.36 Ϯ 1.49 g; weight Ϯ SE; P Յ 0.01; Student’s cut, stained (H&E), and examined for cross-sectional tumor t test). The weight of the resected tumors could partially account area. Representative brain sections of mice treated or not with for this observation. The average s.c. tumor mass taken from noscapine are shown in Fig. 8, A and B, respectively. The

largely replaced normal brain tissue in the tumor center while infiltrating extensively normal brain at the periphery (Fig. 8E). Lumens of large blood vessels were much smaller than in the contralateral tumor-free hemisphere, possibly suggesting com- pression by interstitial pressure (Fig. 8E). Brains of noscapine- treated animals showed clearly reduced numbers of tumor cells. Tumor cells in noscapine-treated tissue extensively infiltrated the normal brain and tended to cluster around blood vessels without altering lumen size. In some cases, small areas of hemocyanin were noted, an observation that usually reflects subsided hemorrhage. Noscapine may have induced death of some rapidly proliferating endothelial cells in tumor vascula- Fig. 6 Time course of s.c. rat C6 glioma tumor growth. Palpable ture, leading to vessel leakage. To try to quantify the difference tumors were established 6 days after injecting 1 ϫ 106 rat C6 glioma in tumor burden between both groups while accounting for the cells s.c. in mice. Mice were treated beginning on day 6 with 300 mg/kg intermixing of normal and tumor cells, we used digital imaging. F noscapine in acidified water by gavage daily for 15 days ( ), whereas Cross-sectional tumor area was determined for each 5-␮m slice vehicle-treated animals received acidified water alone by gavage (ࡗ). Tumor volume shown is Ϯ SE. Day 21, P Յ 0.01 (Student’s t test). and representative striatal sections from vehicle- and noscapine- treated animals are shown (Fig. 8, C and D, blue). Using three-dimensional image analysis reconstruction of brain tumor volume, we found that 15 days of noscapine treatment signifi- sections revealed extensive migration of tumor cells (identified cantly inhibited intracranial brain tumor growth by 78% (Fig. by their darker staining) in the injected hemisphere in both 8G, P Յ 0.01; Student’s t test). In addition, we found a trend groups (Fig. 8, A and B, and insets E and F). Brains of untreated toward increased brain weight in vehicle-treated animals com- animals showed a dense twirling pattern of tumor cells that pared with noscapine-treated animals receiving intracranial tu-

Fig. 7 Rat C6 glioma cells were stereotactically implanted into the striatum. A, striatal section depicting india ink stereotactically injected using the coordinates shown. Rat C6 glioma cells (1 ϫ 103) in serum- free DMEM (vehicle solution) or vehicle solution alone were precisely implanted into the striatum of nude mice using a 10-␮l Hamilton syringe. AP, anterior-posterior; ML, medial- lateral; DV, dorsal-ventral. B, after 15 days of 300 mg/kg noscapine treatment in vehicle solution (dH2O; pH 4.5) or vehicle solution alone administered by gavage, animals were euthanized and perfused, and brains were removed and cut into 1-mm-thick macrosections as shown here.

Fig. 8 Noscapine significantly inhibits the growth of an intracranial glioma model. C6 glioma cells (103) were stereotactically injected into the striatum of nude mice. Animals received noscapine or vehicle solution daily beginning on day 6 and were sacrificed on day 21. A-B, macrosections were inde- pendently embedded in paraffin blocks, and the first 5-␮m section was cut and stained with H&E to determine the cross-sectional tumor area for each section. Repre- sentative H&E-stained striatal 5-␮m sections of vehicle-treated (A) and noscapine-treated (B) animals. Cross-sectional area was determined using an autoscan tool that uses an algorithm to select the target region based on intensity difference between target (darkly stained tumor cells) and background (light pink regions) omit- ting normal tissue and empty spaces (described in detail in “Ma- terials and Methods”). The tumor regions obtained for representative untreated (C) and noscapine (D) sections using the autoscan tool are depicted in blue. E and F, a magnification of the region shown in blue for an untreated (E) and noscapine-treated (F) animal. G, daily noscapine treatment for 15 days resulted in a significant 78% inhibition of rat C6 glioma intracranial tumor growth (o) when compared with vehicle-treated an- P Յ 0.05 (Student’s ,ء .(imals (f t test). Bar ϭ 150 ␮m.

mors (median brain weight, 0.50 Ϯ 0.06 and 0.42 Ϯ 0.09 g for intracranial tumor models suggest that noscapine may be effec- untreated and noscapine-treated, respectively, Ϯ SE). This is tive for the management of some types of gliomas. suggestive of increased brain density as a result of the tumor Toxicity Evaluation. Given the efficiency of noscapine tissue. The striking inhibition of tumorigenicity observed in the to reduce tumor growth, the next concern was to examine its

potential toxicity in a variety of tissues. Extending our previous tissues such as spleen, duodenum, and liver as revealed by findings (22), we show that noscapine had no apparent systemic histopathology (Fig. 9, A–C). Given that the principal toxicity of toxicity, even in animals carrying a heavy tumor burden and existing microtubule-targeting agents is peripheral neuropathy, brain tumors known to cause overall anergy (30). Treated ani- we examined peripheral motor and sensory nerves for evidence mals did not show any signs of behavioral or neurological of neuropathy. We did not find evidence of either tubulo- deficit and were equally active as untreated animals and gained vessicular accumulations, as may be seen with impaired axonal weight. However, unexplained blood vessel dilation in noscap- transport, or axonal degeneration in either sensory or motor fibers ine-treated brain tissue was observed in both the tumor-infil- (Fig. 10A). These types of pathological changes have been reported trated and contralateral hemispheres compared with untreated with other agents that disrupt microtubule function. The absence of tissue (Fig. 8F). such pathology suggests that noscapine may be less toxic to pe- Hematological toxicity was absent as determined by com- ripheral nerves than other reported tubulin-binding agents. plete blood count (Fig. 9B). No significant toxic side effects To further evaluate any potential toxic effects of noscapine could be detected by histopathology in sites of rapidly dividing on peripheral nerves, we examined dorsal root ganglion cultures

Fig. 9 Daily noscapine treatment (300 mg/kg) does not induce pathological abnormalities in tissues with frequent cell proliferation. A, representative micrographs showing H&E- stained 10-␮m-thick sections of duodenum, spleen, and liver from noscapine-treated and vehicle-treated groups of mice. No histopathological differences were noted in these tissues. B, complete blood count analysis of tumor-bearing mice treated (red bars) and untreated (blue bars) with noscapine. No significant differences could be detected between the two groups in the WBC count (WBC), lymphocytes (LY), RBC count (RBC), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), neutro- phils (NE), monocytes (MO), eosinophils (EO), and basophils (BA). C, comparison of distinct WBC populations found in the bone marrow of noscapine- treated or untreated animals (analyzed by flow cytometry using specific lineage markers). No difference could be detected in the population of B cells, T cells, macrophages (Mac), and granulocytes (Gran) between the noscapine-treated (o) and the vehicle-treated f groups. Bar ϭ 150 ␮m.

Fig. 10 Daily oral noscapine treatment results in minimal evidence of peripheral neuropathy. A, representative ventral root sections stained with toluidine blue from vehicle- and noscapine-treated animals. B, cultured dorsal root ganglion cells (DRG) in the absence and presence of noscapine. Total axonal length and the dorsal root ganglion cell halo area were the quantitative parame- ters used to measure neurotoxicity. Dorsal root ganglion cells were cultured for 5 days and then incubated with noscapine for up to 10 days. Percent change in axonal length (C) and percent change of dorsal root ganglion cell halo area (D) after 0 ␮M (f), 25 ␮M (o), 50 ␮M (1), or 250 ␮M (2) noscapine treatment for 4, 8, or 10 days are shown.

in the presence or absence of noscapine (Fig. 10, B–D). Cultures might be defective through alternative means such as p14ARF exposed to 25 and 50 ␮M noscapine for 10 days demonstrated deletion, a common feature in gliomas (42, 43, 44). slowing of growth rate compared with control cultures (Fig. 10, Our observation that noscapine crosses an experimental C and D). Exposure to 250 ␮M, however, caused axonal degener- blood-brain barrier efficiently led us to study whether noscapine ation as measured by progressive reduction in axonal length and could inhibit the tumorigenicity of the rapidly dividing rat C6 reduction of dorsal root ganglion area. These types of changes are glioma cell line in vivo. We found that noscapine showed greater typical of those seen with exposure to vincristine or Taxol (31, 32). than 78% inhibition of the growth of intracranial glioma in immunocompromised mice. Histopathology of noscapine- treated brain tissue revealed a dramatically reduced number of DISCUSSION malignant cells and an increase of dilated blood vessels sur- Antineoplastic agents that interact with microtubules rep- rounded by a layer of neoplastic cells. In contrast, we observed resent an important group of drugs that disrupt mitosis and a massive infiltration of tumor growth and an absence of dilated particularly mitotic spindle activity by interfering with micro- vessels in vehicle-treated animals. Although noscapine treat- tubule dynamics. Microtubule-targeting drugs currently in use ment resulted in a marked reduction of tumor cells and tumor either promote excessive stability of microtubules, such as the infiltration, there remains a significant need for additional treat- taxane family, or induce depolymerization of microtubules like ments that will target residual infiltrated tumor cells. Additional the Vinca alkaloids (33). Our prior results suggest that the most studies are warranted to examine whether noscapine will prove prominent effect of noscapine is on microtubule dynamics, equally efficient in the treatment of mice carrying human tumors significantly enhancing the percentage of time microtubules that exhibit a slower mitotic rate or for the treatment of spon- spend idle or in a paused state (22). In this study, we show that taneously occurring gliomas in transgenic mice. noscapine significantly reduces the viability of rat C6 glioma The use of any chemotherapeutic agent that affects micro- cells at doses that do not induce death in primary mouse glial tubule structure or dynamics raises a concern of neurotoxicity, cells. We cannot exclude that the dose calculations and com- particularly in regard to the peripheral nervous system. These parisons drawn between the control mouse glial cells and rat C6 types of drugs are thought to disrupt normal axonal transport, glioma cells could vary slightly given potential species-specific leading to axonal degeneration and clinical symptoms of numb- sensitivities to drugs. A significantly greater number of primary ness and/or weakness (45). It is encouraging that doses of glial cells compared with glioma cells arrested in mitosis after noscapine that show efficacy against brain tumors did not cause noscapine treatment. Mitotic C6 glioma cells but not normal any overt pathological changes in the peripheral nervous system. Additionally, sensory neurites died only with prolonged expo- glial cells became polyploid after nuclear endoreplication. Mi- sure to high doses of noscapine. There are no human reports of totic cells showed abnormal spindle formation with excessive peripheral neuropathy with the use of low dose noscapine as an and misaligned chromosomes leading to multinucleated cells. antitussive, however, the assessment of neurotoxicity in the Primary cells arrested in G -M without enhanced DNA accu- 2 setting of treatment for brain tumors requires human clinical mulation, whereas treated glioblastoma cells escaped mitotic trials. arrest and accumulated up to 16N DNA content by entering We observed blood vessel dilation in noscapine-treated successive rounds of DNA synthesis in the absence of cell tissue in both the tumor-infiltrated and contralateral brain hemi- division. Our data suggest that C6 glioma cells may have defi- spheres. Additional studies are warranted to examine whether cient G -S and/or mitotic checkpoints, accounting for the en- 1 dilation is present in other organs, to determine whether the hanced DNA content and abnormal mitoses observed. dilation observed relates to the antitussive effects of noscapine, Because cell cycle checkpoint mechanisms in tumor cells and to assess whether the effects observed constitute a clinical are frequently faulty (34–36), cancer cells may be more suscep- risk at the noscapine doses required to achieve antitumor effects. tible than normal cells to noscapine. Our data support the Noscapine only affects microtubule dynamics (22) rather hypothesis that transformed cells proceed improperly through than changing the net equilibrium between the monomer and the the cell cycle resulting in abnormal mitoses and ultimately polymer distribution of tubulin within the cell (23). Normal cells undergo cell death. Evidence of apoptosis was not observed in with intact checkpoint proteins could conceivably tolerate the these experiments. Abrogation of the G1-S checkpoint is a relatively less disruptive microtubule effects of noscapine com- frequent event in the development of gliomas (4–6), and this is pared with other known antimitotic agents. Normal microtubule known to cause failure of arrest of division (continued replica- morphology is retained in nontumor cells after noscapine expo- tion) in response to treatment with microtubule-targeting drugs sure, and cells resume cell division upon noscapine removal. (37). p53 interacts with the centrosome and regulates centro- Upon in vivo treatment, noscapine levels rise only transiently in some duplication (38). p53 prevents cell cycle progression when plasma; pharmacokinetic studies in mice and humans reveal spindle assembly is blocked by antimicrotubule agents (39), and peak concentration at 3 h after oral ingestion and a relatively fast abnormal centrosome amplification and unbalanced chromo- clearance thereafter. This suggests that normal cells likely re- some segregation are observed in p53 null fibroblasts (40). sume cell division after the noscapine concentration decreases Consistent with these findings, p53 was found to prevent hy- below the threshold level in a few hours (27). In support of this perploidy in human glioma cells exposed to nocodazole (41). hypothesis, BrdUrd measurements in duodenum, spleen, and Whether a similar mechanism operated in C6 glioma cells in liver did not show changes in cell division rates between treated response to noscapine is unclear. C6 glioma cells have been and untreated animals (data not shown). These properties of reported to contain wild-type TP53 alleles, but the p53 pathway noscapine might explain the absence of toxicity at sites of

normally dividing tissue or in peripheral nerves observed in our CDK4 genes have frequent mutations of the retinoblastoma gene. On- study. cogene 1996;13:1065–72. Noscapine should be further tested in humans to confirm a 17. Wade A, editor. Martindale, the extra pharmacopoeia. 27th ed. positive adverse event profile and to examine its ability to London: The Pharmaceutical Press; 1977. inhibit the growth of the subset of central nervous system 18. Balint G, Rabloczky G. Antitussive effect of autonomic drugs. Acta tumors that show rapid proliferation rates such as glioblastoma. Physiol Acad Sci Hung 1968;33:99–109. Perhaps, this could best be achieved in conjunction with other 19. Mourey RJ, Dawson TM, Barrow RK, Enna AE, Snyder SH. [3H]noscapine binding sites in brain: relationship to indoleamines and therapies because noscapine alone did not eradicate the tumor the phosphoinositide and adenylyl cyclase messenger systems. Mol type tested in this study. 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Clin Cancer Res 2004;10:5187-5201.

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